Nematicidal compositions and methods of using them

ABSTRACT

There is disclosed method of killing nematodes comprising the step of applying an effective amount of a nematicidal composition comprising a terpene component and compositions suitable for use in the method. The terpene component is preferably in association with water, either as a solution or a suspension. An excipient may also be included, which is suitably hollow glucan particles which encapsulate the terpene component.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 15/458,197 filed Mar. 14, 2017, which is acontinuation application of U.S. patent application Ser. No. 10/586,597filed Apr. 4, 2008, which is a U.S. National Phase patent application ofP.C.T. International Application No. PCT/GB2005/000240 filed Jan. 24,2005, which claims priority to U.S. Provisional Patent Application No.60/538,627 filed Jan. 23, 2004 and No. 60/572,804 filed May 20, 2004,each of which is incorporated by reference in its entirety.

The present invention relates to nematicidal compositions comprising aterpene component, and to methods of killing nematodes by administrationof a nematicidal composition comprising a terpene component.

Nematodes (Kingdom: Animalia, Phylum: Nematoda) are microscopic roundworms. They can generally be described as aquatic, triploblastic,unsegmented, bilaterally symmetrical roundworms, that are colourless,transparent, usually bisexual, and worm-shaped (vermiform), althoughsome can become swollen (pyroform). It is suggested that nematodes arethe most abundant form of animal life and only about 3% of nematodespecies have been studied in detail.

Many nematodes are obligate parasites and a number of species constitutea significant problem in agriculture. It has been suggested that annualcrop loss estimates caused by plant parasitic nematodes are roughly $80billion worldwide, with $8 billion in the USA. Nematodes are a seriouspest and methods to control their parasitic activities are an importantfeature in maximising crop production in modern intensive agriculture.

There are approximately 197 genera and 4300 species of nematodephytoparasites. Plant parasitic nematodes feed on the roots or shoots ofplants.

The nematodes can be ectoparasites (i.e. feed on the exterior of aplant) or endoparasites (i.e. live/feed inside the host) and can bemigratory or sedentary.

Some of the most significant of the plant parastitic nematodes are:

Genus; Common Name

Meloidogyne; Root-knot nematode

Pratylenchus; Lesion nematode

Heterodera; Cyst nematode

Globodera; Cyst nematode

Ditylenchus; Stem and bulb nematode

Tylenchulus; Citrus nematode

Xiphinema; Dagger nematode

Radopholus; Burrowing nematode

Rotylenchulus; Reniform nematode

Helicotylenchus; Spiral nematode

Belonolaimus; Sting nematode

Nematodes are not just parasitic to plants but a number of species areparasitic to animals, both vertebrate and invertebrate. Around 50species attack humans and these include Hookworm (Anclyostoma),Strongylids (Strongylus), Pinworm (Enterolobius), Trichinosis(Trichina), Elephantitis (Wuchereria), Heartworm (Dirofilaria), andAscarids (Ascaris).

It should be noted however that not all nematodes inhabiting soil arephyto-parasitic. A number of saprophagous nematodes exist which do notharm plants, and indeed may actually exist in a symbiotic relationshipwith plants.

The current procedure for the elimination of nematodes in agricultureinvolves treating the soil with methyl bromide (MB). MB essentiallysterilises the soil and provides effective control of a wide range ofsoil-borne pathogens and pests, including fungi, bacteria, nematodes,insects, mites, weeds and parasitic plants. However, MB has asignificant negative impact on the environment.

Problems associated with MB include:

-   -   Eradication of the beneficial soil microflora and microfauna,        resulting in elimination of natural biological control and        resurgence of secondary pests and diseases. The “biological        vacuum” created by the use of potent biocides, such as MB,        results in rapid re-infestation of treated soils.    -   Toxic side-effects on humans, plants (phytotoxicity) and other        non-target organisms. This has safety implications with regard        to handling MB as any contact with the user would be harmful.        There are therefore also major expenses involved with specialist        equipment, training and other precautions involved with ensuring        that MB is used, handled and transported safely.    -   MB is associated with the depletion of the ozone layer.    -   Pollution of the environment, including soil, water and the        atmosphere. MB is, in particular, a major pollutant of        underground water.    -   Pesticide residues in agricultural products, creating health        risks for consumers and major obstacles to the international        agricultural trade. Soil fumigation with MB is known to leave        bromine residues in the soil which can be taken up by, and        accumulate, in plants. Problems with bromine residues in leafy        vegetables, such as lettuce, are quite common. Indeed, in grape        producing regions the use of MB is not permitted due to its        health implications.

For the reasons mentioned above, inter alia, the production and use ofMB is being phased out on a global scale. Under the Montreal Protocol1991, MB use is to be phased out by 2005 in the E.U. and other developedcountries, and by 2015 in the developing countries. There is therefore aneed to identify suitable alternative solutions for managing soil-bornepathogens, in particular nematodes.

The inventor has surprisingly found that terpenes are effective inkilling nematodes.

Terpenes are widespread in nature, mainly in plants as constituents ofessential oils. Their building block is the hydrocarbon isoprene(C₅H₈)_(n).

Terpenes are classified as generally regarded as safe (GRAS) by theEnvironmental Protection Agency (EPA) in the USA and have been used inthe flavour and fragrance industries.

Terpenes have been found to be effective and nontoxic dietary antitumoragents which act through a variety of mechanisms of action (Crowell andGould, 1994—Crit Rev Oncog 5(1): 1-22; and Crowell et al., 1996—Adv ExpMed Biol 401: 131-136). Terpenes, i.e. geraniol, tocotrienol, perillylalcohol, b-ionone and d-limonene, suppress hepatic HMG-COA reductaseactivity, a rate limiting step in cholesterol synthesis, and modestlylower cholesterol levels in animals (Elson and Yu, 1994—J Nutr. 124:607-614). D-limonene and geraniol reduced mammary tumors (Elegbede etal., 1984—Carcinogenesis 5(5): 661-664; and Elegbede et al., 1986—J NatlCancer Inst 76(2): 323-325; and Karlson et al., 1996—Anticancer Drugs7(4): 422-429) and suppressed the growth of transplanted tumors (Yu etal., 1995—J Agri Food Chem 43: 2144-2147).

Terpenes have also been found to inhibit the in-vitro growth of bacteriaand fungi (Chaumont and Leger, 1992—Ann Pharm Fr 50(3): 156-166; Moleyarand Narasimham, 1992—Int J Food Microbiol 16(4): 337-342; and Pattnaik,et al. 1997—Microbios 89(358): 39-46) and some internal and externalparasites (Hooser, et al., 1986—J Am Vet Med Assoc 189(8): 905-908).Geraniol was found to inhibit growth of Candida albicans andSaccharomyces cerevisiae strains by enhancing the rate of potassiumleakage and disrupting membrane fluidity (Bard et al., 1988—Lipids23(6): 534-538). B-ionone has antifungal activity which was determinedby inhibition of spore germination, and growth inhibition in agar(Mikhlin et al., 1983—Prikl Biokhim Mikrobiol. 19: 795-803; and Salt etal., 1986—Adam Physiol Molec Plant Path 28: 287-297). Teprenone(geranylgeranylacetone) has an antibacterial effect on H. pylori (Ishii,1993—Int J Med Microbiol Virol Parasitol Infect Dis 280(1-2): 239-243).Solutions of 11 different terpenes were effective in inhibiting thegrowth of pathogenic bacteria in in-vitro tests; levels ranging between100 ppm and 1000 ppm were effective. The terpenes were diluted in waterwith 1% polysorbate 20 (Kim et al., 1995—J Agric Food Chem 43:2839-2845). Diterpenes, i.e. trichorabdal A (from R. trichocarpa) hasshown a very strong antibacterial effect against H. pylori (Kadota etal., 1997—Zentralblatt fur Bakteriologie. 286(1):63-7).

Rosanol, a commercial product with 1% rose oil, has been shown toinhibit the growth of several bacteria (Pseudomonas, Staphylococcus, E.coli and H pylori). Geraniol is the active component (75%) of rose oil.

In U.S. Pat. Nos. 5,977,186 and 6,130,253, methods of using terpenes tokill lice are disclosed.

In International Patent Application published under WO 03/020024, by thepresent inventor, methods of using terpenes to prevent and treatinfections plants by bacteria, phytoplasmas, mycoplasmas or fungi aredisclosed.

There may be different modes of action of terpenes againstmicroorganisms; they could (1) interfere with the phospholipid bilayerof the cell membrane, (2) impair a variety of enzyme systems(HMG-reductase), and (3) destroy or inactivate genetic material. It isbelieved that due to the modes of action of terpenes being so basic,e.g., blocking of cholesterol, that infective agents will not be able tobuild a resistance to terpenes.

There are, however, a number of drawbacks to the use of terpenes. Theseinclude:

-   -   Terpenes are liquids which can make them difficult to handle and        unsuitable for certain purposes.    -   Terpenes are not very miscible with water, and it generally        requires the use of detergents, surfactants or other emulsifiers        to prepare aqueous emulsions. A stable solution can, however, be        obtained by mixing the terpenes under high shear.    -   Dry powder terpene formulations generally only contain a low        percentage w/w of terpenes.    -   Terpenes are prone to oxidation in aqueous emulsion systems,        which make long term storage a problem.

There are limitations to the current techniques of spray coating,extrusion, coacervation, molecular encapsulation, and spraydrying/cooling to provide ingredient delivery systems.

Yeast cell walls are derived from yeast cells and are composed of theinsoluble biopolymers β-1,3-glucan, β-1,6-glucan, mannan and chitin.They are typically 2-4 micron in diameter microspheres with a shell wallthat is only 0.2-0.3 micron thick surrounding an open cavity. Thismaterial has considerable liquid holding capacity, typically absorbing5-25 times its weight in liquid. The shell is sufficiently porous thatpayloads up to 150,000 Daltons in size can pass through the outer glucanshell and be absorbed into the hollow cavity of the spherical particle.Yeast cell walls have several unique properties, including heatstability (e.g. to 121° C.), shear stability, pH stability (e.g. pH2-12), and at high concentrations they do not build significantviscosity. In addition to its physical properties this compositioncontains the natural and healthy dietary fibres that delivercardiovascular and immunopotentiation health benefits.

Yeast cell walls are prepared from yeast cells by the extraction andpurification of the insoluble particulate fraction from the solublecomponents of the yeast cell. The fungal cell walls can be produced fromthe insoluble byproduct of yeast extract manufacture. Further, the yeastcells can be treated with an aqueous hydroxide solution, withoutdisrupting the yeast cell walls, which digests the protein andintracellular portion of the cell, leaving the yeast cell wall componentdevoid of significant protein contamination, and having substantiallythe unaltered cell wall structure of β(1-6) and β(1-3) linked glucans. Amore detailed description of whole glucan particles and the process ofpreparing them is described by Jamas et al. in U.S. Pat. No. 4,810,646and in co-pending patent applications U.S. Pat. No. 166,929, U.S. Pat.No. 297,752 and U.S. Pat. No. 297,982. U.S. Pat. No. 6,242,594, assignedto Novogen Research Pty Ltd., describes a method of preparing yeastglucan particles by alkali extraction, acid extraction and thenextraction with an organic solvent and finally drying. U.S. Pat. No.5,401,727, assigned to AS Biotech-Mackzymal, discloses the methods ofobtaining yeast glucan particles and methods of using them to promoteresistance in aquatic animals and as an adjuvant for vaccinations. U.S.Pat. No. 5,607,677, assigned to Alpha-Beta Technology Inc., disclosesthe use of hollow whole glucan particles as a delivery package andadjuvant for the delivery of a variety of pharmaceutical agents. Theteachings of the abovementioned patents and applications areincorporated herein by reference.

According to the present invention there is provided a method of killingnematodes, said method comprising the step of applying an effectiveamount of a nematicidal composition comprising a terpene component.Preferred features of the nematicidal composition are described below.

The terpene component may comprise a single terpene or a mixture ofterpenes.

The list of terpenes which are exempted from US regulations found in EPAregulation 40 C. F. R. Part 152 is incorporated herein by reference inits entirety.

Preferably the terpene component comprises one or more terpenes selectedfrom the group comprising citral, pinene, nerol, b-ionone, geraniol,carvacrol, eugenol, carvone, terpeniol, anethole, camphor, menthol,limonene, nerolidol, farnesol, phytol, carotene (vitamin A), squalene,thymol, tocotrienol, perillyl alcohol, borneol, myrcene, simene, carene,terpenene and linalool.

It should also be noted that terpenes are also known by their extract oressential oil names, e.g. lemongrass oil (contains citral).

A suitable terpene component may comprise, for example:

-   -   100% citral;    -   50% citral and 50% b-ionone;    -   50% citral and 50% a-terpineol;    -   50% d-limonene and 50% b-ionone; or    -   50% a-terpineol and 50% b-ionone.

It has been found that compositions comprising citral are particularlyeffective at killing nematodes. Therefore it is preferred that thenematicidal composition of the present invention comprises citral.

It is highly preferable that all compounds present in the nematicidalcomposition are classified as generally regarded as safe (GRAS).

The term “terpene” as used herein refers not only to terpenes of formula(C₅H₈)_(n), but also encompasses terpene derivatives, such as terpenealdehydes. In addition, reference to a single name of a compound willencompass the various isomers of that compound. For example, the termcitral includes the cis-isomer citral-a (or geranial) and thetrans-isomer citral-b (or neral).

In a preferred embodiment the nematicidal composition comprises aterpene component and water. The terpene component may be in solution inthe water. Alternatively the nematicidal composition may comprise asurfactant which holds the terpene in suspension in the water. Suitablesurfactants include, sodium lauryl sulphate, polysorbate 20, polysorbate80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglycerylmonooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate,triglycerol monostearate, TWEEN, Tween 80, SPAN 20, SPAN 40, SPAN 60,SPAN 80, Brig 30 or mixtures thereof. Sodium lauryl sulphate is apreferred surfactant due to its recognised safety.

In one embodiment of the invention the nematicidal composition has a pHof less than 7, suitably a pH from around 3 to less than 7, andpreferably a pH from around 3 to around 5. Where the nematicidalcomposition has a pH below 7 the nematicidal activity of the compositiondoes not decrease over time compared to a composition having a pH over7.

Suitably the nematicidal composition comprises the terpene component ata concentration from about 125 to about 2000 ppm in water, preferablyfrom about 250 to about 1000 ppm. A terpene component concentration fromabout 500 to about 2000 ppm may be preferred if higher kill rates aredesired.

In one embodiment of the invention the terpene component is provided ata concentration at which parasitic nematodes are killed selectively overnon-parasitic nematodes. Suitably the parasitic nematodes are root-knotnematodes and the non-parasitic nematodes are Saprophagous nematodes.

Suitable concentrations include from 250 to 1000 ppm, and preferablyfrom 250 to 750 ppm.

The nematicidal composition may also comprise an excipient. Theexcipient may suitably comprise a liposome. Certain excipients mayaugment the action of the terpene component by, for example, increasingits longevity of action or by increasing its capacity to contact andinteract with nematodes.

A particularly preferred excipient is hollow glucan particles. The term“hollow glucan particle” as used herein includes any hollow particlecomprising glucan as a structural component. Thus, in particular, theterm includes yeast cell walls (in purified or crude forms) or otherhollow glucan particles, which may be hollow whole glucan particles.

It has been found that terpenes can be taken up and stably encapsulatedwithin hollow glucan particles.

According to a further aspect of the present invention there is provideda method of killing nematodes, said method comprising the step ofapplying an effective amount of a nematicidal composition comprising ahollow glucan particle encapsulating a terpene component.

Nematicidal compositions comprising a hollow glucan particleencapsulating a terpene component can provide the following advantages:

-   -   maximise terpene payload;    -   minimise unencapsulated payload;    -   control payload stability;    -   control payload release kinetics;    -   creation of a solid form of a liquid terpene to increase the        mass and uniformity;    -   simplify handling and application of terpenes; and    -   mask the smell and taste of the terpene.

Preferably the hollow glucan particles are yeast cell walls. Yeast cellwalls are preparations of yeast cells that retain the three-dimensionalstructure of the yeast cell from which they are derived. Thus they havea hollow structure which allows the terpene component to be encapsulatedwithin the yeast cell walls. The yeast walls may suitably be derivedfrom Baker's yeast cells (available from Sigma Chemical Corp., St.Louis, Mo.).

Alternative particles are those known by the trade names SAF-Mannan (SAFAgri, Minneapolis, Minn.) and Nutrex (Sensient Technologies, Milwaukee,Wis.). These are hollow glucan particles that are the insoluble wastestream from the yeast extract manufacturing process. During theproduction of yeast extracts the soluble components of partiallyautolyzed yeast cells are removed and the insoluble residue is asuitable material for terpene loading. These hollow glucan particles are˜25-35% glucan w/w. A key attribute of these materials are that theyare >10% lipid w/w and are very effective at absorbing terpenes. Inaddition, as a waste stream product they are a relatively cheap costsource of hollow glucan particles.

Alternative hollow glucan particles which have higher purity are thoseproduced by Nutricepts (Nutricepts Inc., Burnsville, Minn.) and ASABiotech. These particles have been alkali extracted, which removesadditional intracellular components as well as removes the outermannoprotein layer of the cell wall yielding a particle of 50-65% glucanw/w. Since alkali extraction saponifies some of the lipids theseparticles are less effective at absorbing terpenes. They are alsosignificantly more expensive and hence these materials are preferredparticles.

Higher purity hollow glucan particles are the WGP particles fromBiopolymer Engineering. These particles are acid extracted removingadditional yeast components yielding a product 75-85% glucan w/w. Theyare even more expensive than the Nutricepts and ASA Biotech particlesand the lower lipid content results in poor loading with terpenes.

Very high purity hollow glucan particles are WGP from Alpha-betaTechnology, Inc. (Worcester, Mass.) and microparticulate glucan fromNovogen (Stamford, Conn.). These particles are organic solvent extractedremoving residual lipids and are >90% glucan w/w.

Of all of the materials tested so far, these particles absorbed theleast terpenes.

Situations may, however, be envisaged where a high purity glucanparticle is required, for example, where tight control over possiblecontaminants is required. In these instances the higher purity particleswould be preferred over the more crude products, despite their poorerterpene loading characteristics.

Preferably the hollow glucan particles have a slight lipid content. Aslight lipid content can increase the ability of the hollow glucanparticle to encapsulate the terpene component. Preferably the lipidcontent of the hollow glucan particles is greater than 5% w/w, morepreferably greater than 10% w/w.

For encapsulation into a hollow glucan particle the terpene component ofthe present invention can optionally be associated with a surfactant.The surfactant can be non-ionic, cationic, or anionic. Examples ofsuitable surfactants include sodium lauryl sulphate, polysorbate 20,polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester,polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycoldicaprilate, triglycerol monostearate, Tween®, Tween 80, Span® 20, Span®40, Span® 60, Span® 80, Brig 30 or mixtures thereof. The surfactant actsto hold the terpene component in an emulsion and also assistsencapsulation of the terpene component into the hollow glucan particle.

The nematicidal composition of the invention can comprise hollow glucanparticles encapsulating a terpene component which comprise 1 to 99% byvolume terpene component, 0 to 99% by volume surfactant and 1 to 99%hollow glucan particles. More specifically the hollow glucan particlesencapsulating a terpene component can comprise from about 10% to about67% w/w terpene component, about 0.1-10% surfactant and about 40-90%hollow glucan particles. A stable suspension of hollow glucan particlesincorporating citral of 25 ppt citral can be made.

Suitably a nematicidal composition comprises from about 500 to about10,000 ppm hollow glucan particles, where the particles contain fromabout 1 to about 67% terpene component. Preferably the nematicidalcomposition comprises from about 1000 to about 2000 ppm hollow glucanparticles, where the particles contain from about 10 to about 50%terpene component.

The method is particularly suited to killing nematodes in soil,especially in soil used for agricultural or horticultural purposes. Sucha method involves administering a nematicidal composition comprising aterpene component to at least a portion of, preferably all of, the soilto be treated.

Optionally the application of the nematicidal composition may berepeated. This may be necessary in some cases to ensure effectivekilling of the nematodes present in the portion of soil. The applicationof the nematicidal composition to soil may be carried out in a number ofways, including spraying, irrigation or the like.

In one embodiment the nematicidal composition used in the method of thepresent invention may be formed by mixing the terpene component andwater with sufficient shear to create a solution of the terpene inwater. Terpenes are generally poorly soluble in water, however, withmixing at sufficient shear they can be forced to form a stable solutionin water. An aqueous terpene solution has the advantage that it can betaken up by plants through their roots, whereas an aqueous terpenesuspension cannot.

In an alternative embodiment the nematicidal composition may be formedby adding a surfactant to hold the terpene component in aqueoussuspension. Such a suspension would be useful where it is not necessaryfor the composition to be taken up by the plant, e.g. for treating aninfection with ectoparasitic nematodes.

In an alternative embodiment the present invention further provides amethod of preparing a nematicidal composition comprising hollow glucanparticles encapsulating a terpene component, said method comprising thesteps of;

-   -   a) providing a terpene component;    -   b) providing hollow glucan particles;    -   c) incubating the terpene component with the glucan particles        under suitable conditions for terpene encapsulation; and    -   d) recovering the glucan particles encapsulating the terpene        component.

Optionally the above method can further comprise the step of drying theglucan particles encapsulating the terpene component. Drying may beachieved in a number of ways and mention may be made of freeze drying,fluidised bed drying, drum drying or spray drying, all of which are wellknown processes.

In step a) of the above method, the terpene component is suitablyprovided as a suspension in an aqueous solvent, and optionally in thepresence of a surfactant. Suitably the solvent is water. A suitablesurfactant is Tween-80 (polyoxyethylenesorbitan monooleate) or sodiumlauryl sulphate, and preferably the surfactant is present at aconcentration of about 0.1 to 10% by volume of the total reactionmixture, more preferably about 1%. Alternatively the terpene componentmay be provided as a true solution in a solvent, e.g. water. A truesolution of terpene in water can be obtained by mixing the terpene inwater at high shear until a true solution is obtained. Publication NoWO03/020024 provides further details of forming true solutions ofterpenes in water.

In step b) of the above method, the hollow glucan particles are suitablyprovided as a suspension in water or other suitable liquid. Suitably thesuspension comprises approximately 1 to 1000 mg glucan particles per ml,preferably 200 to 400 mg/ml. Alternatively the hollow glucan particlesmay be provided as a dry powder and added to the terpene-surfactantsuspension.

Alternatively the glucan particles are provided in sufficient liquid tominimally hydrate the particles, but not in significant excess. The term“hydrodynamic volume” (HV) is used to describe the volume of liquidrequired to minimally hydrate the particles. Thus suitably the particlesare provided in between the HV and HV+50% of water. This makes thesubsequent drying step more efficient. Also, where a low volume of wateris used (ie. around HV to HV+50%), it is also possible to extrude thefinished product into pellet or noodle form, which is convenient forfluidised bed drying.

It has been found that the terpene component can become encapsulated bythe hollow glucan particles at room temperature. The rate ofencapsulation is, however, increased at 37° C. but the temperatureshould be kept below the boiling point or denaturing temperature of anycomponent of the composition. Suitable conditions for step c) of theabove method are therefore atmospheric pressure at a temperature of 20to 37° C. Optimisation of the conditions for a particular encapsulationreaction will be a matter of routine experimentation.

The present invention also provides the use of a nematicidal compositioncomprising a terpene component as described above for the exterminationof nematodes, especially nematodes in soils and/or infecting plants.

It will be obvious to one skilled in the art that the nematicidal use ofa composition made entirely of compounds which are GRAS is highlypreferable over the use of prior art toxic compositions. Environmentalconcerns associated with use of the composition will be greatly reducedand there would be no significant problems with accumulation of theproduct in food crops. Additionally, regulatory approval of thecomposition in various jurisdictions would not be as difficult to obtainas for a toxic composition, and indeed may not even be required in someinstances.

Embodiments of the present invention will now be described by way ofexample only, with reference to the figures in which:

FIG. 1 represents a light micrograph of empty yeast cell walls;

FIG. 2 represents a light micrograph of yeast cell walls encapsulatingL-carvone;

FIG. 3 represents a light micrograph of yeast cell walls encapsulatingcitral;

FIG. 4 represents a light micrograph of terpene emulsion;

FIG. 5 represents a light micrograph of yeast cell walls in hydrodynamicvolume (HV) water;

FIG. 6 represents a light micrograph of yeast cell walls encapsulatingterpene in 5 times hydrodynamic volume (HV) of water;

FIG. 7 represents a light micrograph of yeast cell walls encapsulatingterpene in HV of water;

FIG. 8 represents a light micrograph of yeast cell walls encapsulatingterpene in HV plus 5% of water;

FIG. 9 represents a light micrograph of yeast cell walls encapsulatingterpene in HV plus 10% of water;

FIG. 10 represents a light micrograph of yeast cell walls encapsulatingterpene in HV plus 20% of water;

FIG. 11 represents a light micrograph of yeast cell walls encapsulatingterpene in HV plus 30% of water;

FIG. 12 represents a light micrograph of yeast cell walls encapsulatingterpene in HV plus 40% of water.

FIG. 13 represents a light micrograph showing the dispersal of driedhollow glucan particles encapsulating a terpene component and no xanthangum.

FIG. 14 represents a light micrograph as in FIG. 13 where 0.07 g of 1%xanthan gum is included.

FIG. 15 represents a light micrograph as in FIG. 13 where 0.14 g of 1%xanthan gum is included.

FIG. 16 represents a light micrograph as in FIG. 13 where 0.28 g of 1%xanthan gum is included.

FIG. 17 represents a light micrograph as in FIG. 13 where 0.55 g of 1%xanthan gum is included.

FIG. 18 represents a light micrograph as in FIG. 13 where 1.1 g of 1%xanthan gum is included.

FIG. 19 represents a light micrograph as in FIG. 13 where 2.2 g of 1%xanthan gum is included.

FIG. 20 represents a light micrograph as in FIG. 13 where 4.4 g of 1%xanthan gum is included.

EXAMPLE 1 Preparation of a Terpene Emulsion or Suspension Using aSurfactant

A terpene, terpene mixture, or liposome-terpene combination can becombined with a surfactant to form a suspension. The volumetric ratio ofterpenes is generally about 1-99%, and the surfactant volumetric ratiois about 1-50% of the solution/mixture. The terpenes, comprised ofnatural or synthetic terpenes, are added to water. The surfactant,preferably polysorbate 80 or other suitable GRAS surfactant, is added tothe water/terpene mixture and then blended to from a suspension. Citralis a suitable terpene.

EXAMPLE 2 Preparation of a Terpene Solution (without Surfactant)

The solution can be prepared without a surfactant by placing theterpene, e. g. citral, in water and mixing under solution-forming shearconditions until the terpene is in solution.

0.5 ml citral was added to 1 litre water. The citral and water wereblended in a household blender for 30 seconds.

Alternatively, moderate agitation also prepared a solution of citral byshaking by hand for approximately 2-3 minutes.

Greater than about zero ppm to about 1000 ppm of natural or syntheticterpenes such as citral, b-ionone, geraniol, carvone, terpeniol,carvacrol, anethole, or other terpenes with similar properties are addedto water and subjected to a solution-forming shear blending action thatforces the terpene(s) into a true solution. The maximum level ofterpene(s) that can be solubilized varies with each terpene. Examples ofthese levels are shown in Table 1.

TABLE 1 Solution levels for various terpenes. Terpene Solution LevelCitral 1000 ppm Terpeniol 500 ppm b-ionone 500 ppm Geraniol 500 ppmCarvone 500 ppm

EXAMPLE 3 Potency of Solution

Terpenes will break down in the presence of oxygen. The rate at whichthey decay varies for each particular terpene.

Citral is a terpene aldehyde and will decay over a period of days. Twoprotocols are described below which quantify the rate of decay ofcitral.

The following protocol was used to determine the rate of decay of citralin a sealed container:

Test Material

A solution prepared as described in Example 2 containing citral at 1000ppm was prepared in distilled water. This solution was stored in acapped glass vial for the duration of the test.

Procedure

A standard curve was prepared with citral and B-ionone as internalstandard.

At the beginning of the study and weekly for four weeks the 1000 ppmsuspension was analyzed using a gas chromatography procedure. Theconcentration of citral was determined by plotting it on the standardcurve.

The results are shown below in Table 2.

TABLE 2 Stability of citral Percentage of citral remaining Day 1 Week 1Week 2 Week 4 Citral 100 32 27 22 (1000 ppm)

The following protocol was used to determine the rate of decay of citralin a container with a porous lid.

To determine the concentration of citral in water the following protocolwas used.

Test Material

A solution containing citral at 1000 ppm was prepared in distilledwater. This solution was stored in a beaker covered with porous paperfor the duration of the test.

Procedure

A standard curve was prepared with citral and B-ionone as internalstandard.

At the beginning of the study and after a week the 1000 ppm suspensionwas analyzed using a gas chromatography procedure. The concentration ofcitral was determined by plotting it on the standard curve.

The results are shown below in Table 3.

TABLE 3 Stability of citral Percentage of citral remaining Day 1 Week 1Citral 100 21.5% (1000 ppm)

EXAMPLE 4 Extraction of Nematode Eggs from Soil and Counting NematodeNumbers

Extraction of Eggs and Quantification of Soil Populations

The following is an outline of a suitable technique to determine thepopulation densities of soybean cyst nematodes SCN in soil samples,although it would be applicable to other soil nematodes. The procedurehas three stages:

-   -   extracting the cysts from the soil;    -   crushing the cysts to extract the eggs; and,    -   microscopic observation of the suspension of eggs for counting.

Extraction of Cysts from Soil

Cysts of soybean cyst nematode are recovered from soil through acombination of wet-sieving and decanting. The technique is amodification of the Cobb (Cobb, N. A. 1918. Estimating the nemapopulation of soil. U.S. Dept. Agr. Bur. Plant Ind. Agr. Tech. Cir.,1:1-48) sifting and gravity technique.

The procedure is as follows:

-   1. Combine a well mixed 100 cm³ soil sample (approx. ½ cup) in a    bucket with two (2) quarts (2.27 litres) of water.-   2. Break any clumps with your fingers and mix the soil suspension    well for 15 seconds.-   3. Pour the soil suspension through an 8-inch-diameter #20 (850 mm    pore) sieve into another bucket. Briefly rinse the debris caught on    the 20 mesh sieve.-   4. Pour the soil suspension in the second bucket through a #60 (250    mm pore) sieve.-   5. Backwash the debris caught on the 60 mesh screen into a pan.-   6. Repour the suspension through the 60 mesh screen—hold the screen    at an angle to concentrate the cysts and debris.-   7. Backwash into a pan using a minimal (<250 ml) amount of water.-   8. Pour the cysts and debris into a 250 ml beaker. NOTE: Discard the    heavier material that quickly settles to the bottom of the    buckets/pans during the above sieving process.

Extraction of Eggs from the Cysts

The above technique will result in a suspension of SCN cysts, along withorganic debris and sediments similar in size to the cysts. The cysts inthis suspension could be counted using a simple dissecting microscope.Some laboratories that analyze soil for soybean cyst nematode reportresults in the form of cysts per 100 cm³ of soil. Egg content of cystsis highly variable, and will not yield reliable counts of the SCNpopulation in the sample. Therefore, it is preferable if eggs areextracted from the cysts and results are reported back as eggs andsecond stage juveniles (J-2) per 100 cm³ of soil.

The procedure used to extract eggs from cysts is as follows:

-   -   1. Allow cysts/debris to settle for ca 30 minutes in the 250 ml        beakers. Pour off excess water, resuspend sediments and transfer        to 50 ml beakers.    -   2. Allow cysts to settle in the 50 ml beakers.    -   3. Pour off excess water (˜30 ml) and transfer the cyst/debris        suspension to a 55 ml Wheaton Potter-Elvehjen tissue grinder.    -   4. Grind at 7500 RPM for 10 seconds. Rinse pestle into grinding        tube.    -   5. After grinding, pour the suspension in the tube through an        8-inch-diameter #200 (75 mm pore) sieve over a stainless steel        #500 (25 mm pore) sieve.    -   6. Rinse the tube several times with tap water, each time        pouring the contents through the sieves. Discard sediments        caught on the #200 sieve.    -   7. Carefully wash sediments and eggs caught on the #500 sieve        into a clean beaker with as little water as possible.

Counting Eggs with the Nematode Counting Slide:

The volume of the egg suspension should be brought up to exactly 50 mlwith tap water. Fill the chamber of the nematode counting slide with awell-mixed suspension using a pipette. The specially made nematodecounting slides are constructed so that the volume of egg suspensionobserved over the grid is exactly 1 ml. Consequently, simply count thenumber of eggs that appear within the grid of the slide to determine thenumber of eggs per ml of suspension. The total number of eggs in thesample can then be calculated by multiplying the number of eggs per mlby 50.

Sources of Materials and Equipment

Sieves:

-   -   Fisher Scientific, 1600 W. Glenlake Avenue, Itasca, Ill.        60143—(800) 223-9114    -   VWR Scientific, P.O. Box 66929, O'Hare AMF, Chicago, Ill.        60666—(800) 932-5000

Tissue Grinder:

-   -   Fisher Scientific, 1600 W. Glenlake Avenue, Itasca, Ill.        60143—(800) 223-9114

Motorized Stirrer:

The motorized laboratory stirrer is a Talboys Model 101 stirrer. Thisstirrer can be purchased through VWR Scientific or directly throughTalboys Engineering Corporation, South Montrose, Pa. 18843.

Nematode Counting Slides:

The specially made nematode counting slides can be purchased fromAdvanced Equine Products, 5004 228th Avenue S.E., Issaquah, Wash. 98029,(425) 391-1169, FAX (425) 391-6669.

EXAMPLE 5 Effect of Terpenes on Nematode Egg Hatching and JuvenileSurvival

The effect of various terpene containing compositions was assessed inrelation to nematode eggs and juvenile nematodes.

The protocol used was as follows:

The live eggs were treated in the various samples for one hour, rinsed,put back into distilled water and counted 24 hours later. The sampleswere made up as shown in Table 4a:

TABLE 4a Sample Components NM1 10% Tween 80 45% d-limonene 45% b-IononeNM3 10% Tween 80 45% citral 45% b-Ionone NM5 10% Tween 80 45% citral 45%a-terpineol NM6 10% Brig 30 45% a-terpineol 45% b-Ionone NM7 10% Tween80 45% a-terpineol 45% b-Ionone

The results of the protocol are shown below in Table 4b.

TABLE 4b Results Sample Conc. Egg batch Juveniles Designation (%) (%)alive (%) Control — 19 86 NM1 0.5 3 0 0.1 10 19 0.05 17 67 NM3 0.5 2 10.1 5 3 0.05 10 31 NM5 0.5 4 0 0.1 9 16 0.05 16 37 NM6 0.5 11 13 0.1 1736 0.05 16 48 N6 0.5 26 53 0.1 26 58 0.05 15 60 NM7 0.5 13 74 0.1 13 580.05 17 75

Observations: The combinations containing citral (NM3 and NM5) were moreeffective. The Brig surfactant was not as effective as Tween 80. Thealdehyde worked better than the alcohols.

EXAMPLE 6 Effect of Terpenes on Mature Root-Knot, Ring and CitrusNematodes

The effect of various terpene containing compositions was assessed inrelation to Root-Knot nematodes (Meloidogyne), Ring nematodes(Criconemella xenoplax) and Citrus nematodes (Tylenchulussemipenetrans).

The protocol used was as follows:

Nematodes: A single 5 ml volume with pre-counted nematode numbers wasused as the initial inoculum. Nematodes were collected, identified andmaintained from commercial agricultural crops soils. The nematodes werecounted and evaluated for good health for the duration of the study.

Nematicidal compositions: In this protocol the terpene used in thenematicidal composition was citral. The relevant details of the citralused are as follows:

-   Chemical Name: CITRAL-   Common Name: Lemongrass Oil-   Formulation: CITRAL FCC-   Product Trade Name: CITRAL FCC-   Product code: 03-29200-   Source: Penta Manufacturing-   Lot Numbers: 77887-   Type: Liquid-   Carrier: Distilled Water-   Storage Conditions: Ambient indoor room temperature ˜65° F. (28.3°    C.)-   Stability: Insoluble in water above 1,000 ppm.

3 different concentrations of citral were used to assess the efficacy ofterpenes in killing the nematodes. These were untreated control (UTC),500 ppm and maximum soluble terpene concentration (900 ppm). Theterpenes were combined with water as a solution by mixing at a solutionforming shear. The 900 ppm concentration value was not be measured, butestimated at the maximum soluble concentration that can be obtained withdistilled water at 65° F. (28.3° C.). 3 replicates of the 900 ppmconcentration were used (R1, R2 and R3) and one replicate of the 500 ppmconcentration and UTC.

Test mixtures of nematodes and the nematicidal compositions were made upaccording to Table 5.

TABLE 5 Test Mixtures Terpene Added Nematode + Nematode Conc. TerpeneTerpene Treatment Label Vol. ml ppm Vol. Ml Vol. Conc. ppm UTC 5.0 0.05.0 10.0 0.0 1.0 5.0 500.0 15.0 20.0 375.0 R1 5.0 900.0 15.0 20.0 675.0R2 5.0 900.0 15.0 20.0 675.0 R3 5.0 900.0 15.0 20.0 675.0

The terpene and nematode containing water was combined to form a finaldilution volume and maintained in vials between evaluations. Thenematodes were exposed to the terpenes for between 48 to 72 hoursdepending on their survival.

Evaluations: Nematodes were be counted and their appearance assessed bymicroscope. The microscope used for assay provided for only 5 ml to beviewed at one time. Therefore, the 20 ml of total terpene nematodesample water was divided into 4 parts for each assay and recombinedafterwards. The rating of degree of efficacy of the test samples wasdetermined by observing nematode mobility, mortality, and internaldisruption or vacuolation over time.

The results are shown below in Table 6.

TABLE 6 Results Sample I.D. Root-Knot Ring Citrus Meloidogyne CX TS DayTreatment Time Alive Dead alive Dead alive dead (pretreatment reading) 1UTC 11:00am 351 0 357 0 148 0 1 1.0 11:00am 359 0 325 0 119 0 1 20 ml-R111:00am 326 0 264 0 132 0 1 20 ml-R2 11:00am 347 0 260 0 141 0 1 20ml-R3 11:00am 328 0 442 0 137 0 (postreatment readings) 1 UTC 6:00pm 3480 350 0 144 0 1 1.0 6:00pm 355 0 319 0 114 0 1 20 ml-R1 6:00pm 320 0 2580 128 0 1 20 ml-R2 6:00pm 341 0 255 0 139 0 1 20 ml-R3 6:00pm 325 0 4360 134 0 2 UTC 6:00am 344 0 348 0 140 0 2 1.0 6:00am 350 0 312 0 112 0 220 ml-R1 6:00am 140 176 91 0 160 0 2 20 ml-R2 6:00am 168 169 110 141 4684 2 20 ml-R3 6:00am 137 184 181 248 70 59 2 UTC 6:00am 340 0 342 0 1350 2 1.0 6:00am 340 6 304 4 101 8 2 20 ml-R1 6:00am 0 302 0 239 0 109 220 ml-R2 6:00am 0 322 0 236 0 116 2 20 ml-R3 6:00am 0 305 0 402 0 117 3UTC 6:00am 330 3 336 1 126 5 3 1.0 6:00am 189 149 190 108 47 51

There was a small nematode loss from one reading to another due tonematodes hanging up on the sides of dishes and vials. These populationsare usually under 5 nematodes per reading.

Observations:

Day 1—pretreatment readings showed no dead nematodes and the nematodeswere all moving and had no internal disruption or vacuolation.

Day 1—6 pm (20 ml—R1+R2+R3) treatments all appeared to have slowedmovement but they had no internal disruption or vacuolation.

Day 1—6 pm (1.0 and UTC) treatments showed no slowing of movement orinternal disruption or vacuolation.

Day 2—6 am (UTC and 1.0) treatments all appeared normal with no loss ofmovement and no internal disruption or vacuolation.

Day 2—6 am (20 ml—R1+R2+R3) treatments had some dead (dead had nomovement and their internal body structures were highly vacuolated). Theliving nematodes were still moving, although slowly, but no internaldisruption or vacuolation.

Day 2—6 pm (UTC) treatment all appeared normal with no loss of movementand not internal disruption or vacuolation.

Day 2—6 pm (1.0) treatment had some dead. Dead had no movement withinternal disruption and vacuolation. Some of the living had slowedmovement and some did not, but none had any internal disruption orvacuolation.

Day 2—6 pm (20 ml—R1+R2+R3) treatments were all dead with no movementand internal disruption with vacuolation.

Day 3—6 am (UTC) treatments showed a few dead or dyeing nematodes. Theyhad no movement but showed no internal disruption or vacuolation. Therest of the nematodes, listed as alive, still had good movement.

Day 3—6 am (1.0) treatments showed about 50% dead and both internaldisruption and vacuolation. The alive nematodes showed some slowing ofmovement but no internal disruption or vacuolation.

As can be clearly seen from the results, on day two by 6 pm,compositions R1, R2 and R3 had killed all nematodes. This demonstratesthe highly nematicidal properties of compositions R1, R2 and R3 andconsequently the nematicidal properties of citral.

EXAMPLE 7 Effect of Citral Alone and Citral and Thymol on Root-KnotNematode Juveniles

Treatment samples were prepared as follows:

Cital—1 ml citral was added to 400 ml of sterile distilled water andmixed using a household blender for 40 seconds. This was labelled 2500ppm and was diluted to provide test solutions at 500, 250, 125 and 62.5ppm.

Citral and Thymol—1.0 g of thymol was dissolved in 1 ml of citral andblended in 400 ml of water as for citral alone. This was marked 2500 ppmand diluted to provide test solutions at 500, 250, 125 and 62.5 ppm.

Control—Water was used as the control.

Nematode juveniles were collected in water and 0.1 to 0.15 ml added toeach well of a plastic assay plate. 1.0 ml of the test solutions wasadded to each well. Observations were made microscopically after 24 and48 hours as described in Example 4. Dead nematodes adopt a straightposition and do not move when probed with a fine needle. Livingnematodes move in an undulating, wave-like motion.

The results of two experiments are provided below in Tables 7 and 8. Thefigures given are for the percentage of nematodes found to be dead uponmicroscopic examination and are the average of 2 replicates.

TABLE 7 Effect of test solutions of root-knot juveniles after 24 and 48hours Citral and Thymol Cital (ppm) Control Test(ppm) 500 250 125 500250 125 Water 24 h 100 100 100 98 100 100 10 48 h 100 91 50 97 91 24 31

TABLE 8 Effect of test solutions of root-knot juveniles after 24 hours.Citral and Thymol Cital (ppm) Control Test(ppm) 250 125 62.5 250 12562.5 Water 24 h 97 96 94 94 94 98 6

The results demonstrate the ability of citral alone and a citral andthymol mixture to kill nematodes at low concentrations. Kill rates intable 7 after 48 hours were over 90% for both mixtures at 250 ppm and500 ppm concentrations. The 125 ppm concentration showed a lower killrate. The kill rates in Table 8 show high kill rates after 24 hours forconcentration as low as 62.5 ppm.

The mixture of thymol and citral did not show a significant increase inkill rate over citral alone.

The results show that citral is an effective nematicide even at lowconcentrations.

EXAMPLE 8 Effects of Citral on Root-Knot Nematodes vs SarprophagousNematodes

The purpose of this experiment was to demonstrate that citralselectively kills the harmful root-knot nematodes over saprophagusnematodes, which are not harmful, and indeed may be beneficial to theplant and soil. Such selective killing is a surprising effect that meanstreatment with terpenes may kill parasitic nematodes, but not eliminatethe beneficial micro-fauna in the soil.

Aqueous text mixtures comprising 250 ppm citral alone and 250 ppm citraland 10% tween were produced according to the techniques described inExample 7 above. These compositions were then incubated with root-knotand saprophagus nematodes and the kill rate assessed microscopically.Living saprophagus nematodes move rapidly in water. The control used wasthe nematodes in water alone.

The results are provided in Tables 9 and 10 below. The figures given arefor the percentage of nematodes found to be dead upon microscopicexamination and are the average of 2 replicates.

TABLE 9 Nematicidal activity of citral on root-knot nematodes (% dead)Citral + Citral Citral + Citral Tween 80 (250 Tween 80 (250 (250 ppm)ppm) (250 ppm) ppm) Control 24 h 87 87 89 88 17 48 h 100 100 100 100 22

TABLE 10 Nematicidal activity of citral on Saprophagous nematodes (%dead) Citral + Citral Citral + Citral Tween 80 (250 Tween 80 (250 (250ppm) ppm) (250 ppm) ppm) Control 24 h 45 43 51 50 15 48 h 50 50 53 52 19

The results clearly show that citral kills the pathogenic root-knotnematodes at a much higher kill rate than the beneficial saprophagusnematodes. After 48 hrs the kill rate for root-knot nematodes was 100%for all test mixtures, whereas for Saprophagus nematodes it was only50-53%. The results were not significantly effected by the inclusion ofTween 80.

The results demonstrate that terpenes have the ability to selectivelykill pathogenic nematodes whilst allowing beneficial nematodes tosurvive in the soil. This would result in a more healthy soilenvironment post treatment than a treatment which kills the entirenematode population in the soil. Firstly this is because beneficialnematodes would be present in the soil post treatment, and secondlythere would not be a nematode “vacuum” in the soil which could be filledwith pathogenic nematodes or other pathogens.

It could be expected that at a very high concentration of terpene mayresult in a higher kill rate of saprophagus nematodes, thus reducing theselectivity of the treatment. Therefore in use in the field the minimumconcentration that achieves the desired kill rate in root-knot or otherparasitic nematodes may be selected, thus maximising the selectivity.

EXAMPLE 9 Effect of pH on the Nematicidal Activity of Citral ContainingCompositions

The following protocol was performed to assess the affect of pH on testsolutions containing citral.

Solutions were made up of citral at 250, 125 and 62.5 ppmconcentrations. Test solutions of these three concentrations wereprepared at different pHs by adjusting the pH with HCl or NaOH to pH 4,7 and 10.

One batch of test solutions was used immediately and another was leftfor 24 hours before use. The method of administration to the nematodesand counting the kill rate is the same as for previous protocols.

The results are shown below in Tables 11 and 12. The figures given arefor the percentage of nematodes found to be dead upon microscopicexamination and are the average of 2 replicates.

TABLE 11 Effect of fresh citral at three pH levels on root-knotnematodes (% nematodes dead) 250 ppm 125 ppm 62.5 ppm PH 4 7 10 4 7 10 47 10 Water 24 h 75 73 83 31 44 39 48 39 32 21 48 h 73 72 87 50 47 39 5044 45 30

TABLE 12 Effect of one-day old citral at three pH levels on root-knotnematodes (% nematodes dead) 250 ppm 125 ppm 62.5 ppm PH 4 7 10 4 7 10 47 10 water 24 h 90 40 47 27 25 25 40 30 16 10 48 h 90 33 52 31 33 32 2727 21 14

The results demonstrate that, in general, the test solutions loseefficacy if left for one day before use. However, it was observed thatthe citral solutions at the low pH (i.e. 4) did not lose efficacy tosuch an extent and, in fact the 250 ppm sample actually increased inefficacy after being left for a day. At all concentrations tested, thelow pH samples did not demonstrate nearly such a significant a drop ofefficacy after being left when compared to the neutral and high pHcounterparts.

This demonstrates that low pH of citral is beneficial in terms ofretaining the efficacy of citral as a nematocide over time. The reasonsfor this are unclear, but may be the result of stabilising the citraland preventing degradation.

It is therefore clear that adjusting the pH of a citral containingnematicidal composition to be acid (i.e. a pH below 7) would bebeneficial in terms of prolonging its action.

EXAMPLE 10 Comparison of Nematicidal Activity High Purity Citral (98%Pure) with Low Purity Citral (80% Pure)

Citral is commercially available in 2 forms—regular (98% pure) andtechnical (80% pure). The following protocol was carried out todetermine if technical citral is a viable alternative to pure citral.

Compositions of regular and technical ciral at 250 and 125 ppm wereproduced in 1% Tween 80 and incubated with root-knot nematodes a in thesame way as previously described. Observations of the kill rate(percentage dead) were made at 21 and 42 hours.

The results are shown below in Table 13 and are the average of fourreplicates.

TABLE 13 average percentage dead Citral (98% pure) Citral (80% pure) 1%Tween 80 Water Ppm 250 125 250 125 — — 21 h 87 23 89 29 14 7 42 h 87 2296 27 17 18

The results indicate that both regular and technical citral killnematodes effectively at concentrations of 250 ppm. Thus technicalcitral may be used as a cheaper alternative to regular citral.

EXAMPLE 11 Nematicidal Effects of Citral in Soil

The following protocol was carried out to assess the nematicidalproperties of nematodes in soil.

Methodology: Nematodes used for the analysis originated from commercialagricultural crop soils. Species of nematode included root-knot andcitrus. Prior to commencement of each study the nematodes were countedand evaluated for viability. In each experiment soil samples wereinfected with only one species of nematode. Three measured quantities ofsoil (250 g) were placed into large PVC plastic containers.

Soil moisture was assessed by weighing a soil sample and then drying thesample in a drying oven. Soil moisture content was confirmed using a“Hydroscout” instrument. In all cases the moisture content measured byboth methods was within the resolution of the instruments. Bydetermining the water content of the soil it was possible to calculatethe volume of terpene solution which would be diluted when mixed withthe soil.

A series of citral dilutions in water were prepared (500 ppm to 62.5ppm) such that when they were added to the soil samples, they wouldyield the required ratios. These dilutions were by volume not the morecommonly used mass ratios. The reason for using volume dilutions wassimply one of convenience enabling the use of a micropipette or cylinderto measure the terpene. The mass ratio of the ‘in soil’ and ‘in water’solution could be simply calculated by multiplying the ppm of terpene byit's density (0.92 g/ml).

The terpene solution was added to each test tube containing a weighedsample of nematode infected soil. The terpene solution and soil weremixed by inverting the test tube several times. The test tubescontaining the soil and terpene solution were left to stand in racks inthe laboratory for 48 hours-72 hours depending on the survival of theuntreated nematodes. In each experiment a control group was treated withdistilled water. The % mortality (kill) rates in the treatment groupswas compared with the control population.

The nematodes were extracted by “Sieving & mist extraction” (Ayoub, S.M. 1977) prior to being counted.

Criteria for Evaluation: Nematode counts were performed to determine theproportion of nematodes which survived and were killed in each treatmentgroup.

TABLE 14 Pretreatment nematode counts Sample ID Root-Knot Citrus Meannematode 659.25 12,711.75 counts (N = 8)

The results are shown below in Tables 15 and 16.

TABLE 15 Treatment of Root Knot nematodes with terpene solution. Terpeneconcentration No of Replicates Mean % killed 500 ppm 8 67.10 250 ppm 823.66 125 ppm 8 4.34 62.5 ppm  8 18.87 untreated 8 5.71

TABLE 16 Treatment of Citrus nematodes with terpene solution Terpeneconcentration No of Replicates Mean % killed 500 ppm 8 95.53 250 ppm 891.66 125 ppm 8 46.29 62.5 ppm  8 −2.84 untreated 8 13.7

The protocol was repeated, this time using only citral at 500 ppmconcentration. The results are shown below on Table 17 to 19.

TABLE 17 Pretreatment nematode counts Sample ID Root-Knot Citrus Meannematode 1225.25 10755.5 counts (N = 8)

TABLE 18 Treatment of Root-Knot nematodes with terpene solution Terpeneconcentration N Mean % killed 500 ppm 10 99.6

TABLE 19 Treatment of Citrus nematodes with terpene solution Terpeneconcentration N Mean % killed 500 ppm 10 99.9

The experiment was performed once again, this time with the followingchanges:

-   -   Dose range of 125 ppm-750 ppm was used.    -   Glass tubes containing 150 g of soil were used as opposed to PVC        tubes in previous experiments.

The results are shown below in Table 20.

TABLE 20 Treatment of Root Knot nematodes with terpene solution TerpeneMean % concentration N killed 750 ppm 8 99.42 500 ppm 8 100 250 ppm 897.37 125 ppm 8 74.51

The results all show that terpenes are effective nematicides in soil.This supports the data already provided showing that terpenes areeffective nematicides in vitro. Concentrations of terpene as low as 125ppm demonstate strong nematicidal activity in soil, thoughconcentrations of 250 ppm and above showed more consistent high killrates.

EXAMPLE 12 Demonstration of Terpene Loading into Bakers Yeast Particlesand Purified Yeast Glucan Particles

The following protocol was performed to demonstrate that terpenes wouldload into yeast cell walls and other yeast glucan particles.

Emulsions of citral and L-carvone were prepared by mixing 150 μl of theterpene with 100 μl of 10% Tween 80 in water and 250 μl of water.

Baker's yeast particles (YP) or Levacan™ yeast glucan particles (YGP),available from Savory Systems International, Inc., Branchburg, N.J.,were mixed with water to form a 250 mg/ml suspension.

500 μl of the YP or YGP suspension and 250 μl of the terpene emulsionwere mixed together and incubated overnight under constant agitation.500 μl YP or YGP suspension and 500 μl of water were used as a control.The particles were then washed with water until free from externalemulsion. The particle preparations were then frozen and lyophiliseduntil dry.

The particles were then rehydrated and examined under light microscope.The results are shown in FIGS. 1 to 4.

FIG. 1 shows spherical structures with a dark area at their centre,these are empty hollow glucan particles. FIGS. 2 and 3 shows sphericalstructures with a swollen appearance with a light coloured interior,these are particles with terpene encapsulated in the centralcavity—citral in FIG. 2 and L-carvone in FIG. 3. In FIGS. 2 and 3 smallblobs of free terpene can also be seen, e.g. at the top of FIG. 2, justleft of centre. FIG. 4 shows the terpene emulsion as small blebs ofterpene suspended in water.

EXAMPLE 13 Determination of Maximal Citral and L-Carvone Loading Levelsin Baker's Yeast Particles (YP)

The following protocol was performed to determine the maximal amounts ofterpenes that would load into YP.

-   -   L-carvone and citral emulsions were prepared by sonicating 4.5 g        of the terpene with 0.3 ml water.    -   10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80        in 40.5 mls water.    -   YP suspension was prepared by mixing YP with water to form 20        mg/ml suspension.    -   Encapsulation reactions were set up as described in Table 21.

Citral or L-carvone-water emulsion was mixed with YP and Tween 80surfactant overnight at room temperature. Samples were centrifuged at14,000×g for 10 minutes and the appearance of free terpene floating onthe aqueous layer was scored. The results are shown in the right handcolumn labelled free terpene of Table 21.

The expression “free terpene” refers to the visible presence of terpenein the centrifuged reaction mixture. The absence of free terpeneindicates complete absorption of the terpene by the particles. Thehighest volume of terpene absorbed by the particles, as evidenced by theabsence of free terpene, was recorded as the maximal volume of absorbedterpene emulsion.

TABLE 21 20 mg/ml 10% Tween- YP Terpene Vol 80% Free Tube μl Emulsion μlμl Terpene 1 500 — — 500 − 2 500 L-carvone 0.5 500 − 3 500 L-carvone1.65 500 − 4 500 L-carvone 5 495 − 5 500 L-carvone 16.5 483.5 − 6 500L-carvone 50 450 + 7 500 L-carvone 165 335 + 8 500 L-carvone 500 — + 9500 Citral 0.5 500 − 10 500 Citral 1.65 500 − 11 500 Citral 5 495 − 12500 Citral 16.5 483.5 +/− 13 500 Citral 50 450 + 14 500 Citral 165 335 +15 500 Citral 500 — +

As can be seen from the results, YP is capable of absorbing andencapsulating at least 16.5 μl of L-carvone terpene emulsion or at least5 μl of citral emulsion per 10 mg of YP.

EXAMPLE 14 Demonstration of Improved Terpene Loading with Surfactant andDetermination of Optimal Tween-80:Terpene Ratio

The following protocol was performed to demonstrate that the presence ofsurfactant improves terpene loading and to determine the minimum levelof Tween-80 surfactant required for the YP terpene loading reaction.

-   -   L-carvone and citral emulsions were prepared by sonicating 4.5 g        of the terpene with 0.3 ml water.    -   10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80        in 40.5 ml water.    -   Baker's YP suspension was prepared by mixing YP with water to        form 250 mg/ml suspension.

Loading reactions were set up as shown in Table 22 below.

Citral or L-carvone-water emulsion was mixed with YP with 0-10% v/vTween 80 surfactant overnight at room temperature. Samples werecentrifuged at 14,000×g for 10 minutes and the appearance of freeterpene floating on the aqueous layer was scored. The results are shownin the right hand column labelled free terpene of Table 22.

The expression “free terpene” refers to the visible presence of terpenein the centrifuged reaction mixture. The absence of free terpeneindicates complete absorption and encapsulation of the terpene by theYP. The highest volume of terpene absorbed by the YP, as evidenced bythe absence of free terpene, was recorded as the maximal volume ofabsorbed terpene emulsion.

TABLE 22 250 mg/ml YP Terpene Vol 10% Tween-80 Water Free Tube mlEmulsion μl μl μl Terpene 1 500 — — — 500 − 2 500 L-carvone 150 0 350 Sl3 500 L-carvone 150 5 345 Sl 4 500 L-carvone 150 10 340 Sl 5 500L-carvone 150 33 317 Sl 6 500 L-carvone 150 100 250 − 7 500 L-carvone150 200 150 − 8 500 L-carvone 150 350 — − 9 500 L-carvone 400 0 100 ++10 500 L-carvone 400 5 95 ++ 11 500 L-carvone 400 10 90 ++ 12 500L-carvone 400 33 77 ++ 13 500 L-carvone 400 100 — + 14 500 L-carvone 40020 μl 100% 30 + 15 500 Citral 113 0 387 + 16 500 Citral 113 5 382 + 17500 Citral 113 10 377 + 18 500 Citral 113 33 354 Sl 19 500 Citral 113100 287 Sl 20 500 Citral 113 200 187 − 21 500 Citral 113 350 37 − 22 500Citral 250 0 250 ++ 23 500 Citral 250 5 245 ++ 24 500 Citral 250 10 240++ 25 500 Citral 250 33 217 + 26 500 Citral 250 100 150 + 27 500 Citral250 20 μl 100% 230 + Sl = slight

As can be seen from the results a Tween-80 concentration of 1% (i.e. 100μl of 10% Tween-80 in 1000 μl of reaction mixture) is sufficient toallow complete uptake of the terpene in the above reaction. A 2%Tween-80 causes no improvement in results, whereas with a 0.33%concentration free terpene was observed. This indicates that:

-   a) Terpenes are absorbed into YP particles in the absence of a    surfactant, but the presence of surfactant significantly increases    terpene absorption.-   b) A Tween-80 concentration of around 1% is optimum for YP loading    as it ensures proper loading whilst maximising the terpene payload    of the YP particles.

EXAMPLE 15 Determination of Maximal Terpene Loading and Encapsulation atHigh Baker's Yeast Particles (YP) Levels

The following protocol was performed to determine the maximal amounts ofterpenes that would load into YP at high YP levels.

-   -   L-carvone and citral emulsions were prepared by sonicating 4.5 g        of the terpene with 3 ml 1% Tween.    -   5% Tween-80 solution was prepared by sonicating 0.5 g Tween-80        in 9.5 ml water.    -   YP suspension was prepared by mixing YP with water to form 250        mg/ml suspension.    -   Encapsulation reactions were set up as shown in Table 23.

Citral or L-carvone-water emulsion was mixed with YP and Tween 80surfactant overnight at room temperature. Samples were centrifuged at14,000×g for 10 minutes and the appearance of free terpene floating onthe aqueous layer was scored. The results are shown in the right handcolumn labelled free terpene of Table 23.

The expression “free terpene” refers to the visible presence of terpenein the centrifuged reaction mixture. The absence of free terpeneindicates complete absorption of the terpene by the YP. The highestvolume of terpene absorbed by the YP, as evidenced by the absence offree terpene, was recorded as the maximal volume of absorbed terpeneemulsion.

TABLE 23 250 mg/ml 1% Tween- YP Terpene Vol 80% Free Tube μl Emulsion μlμl Terpene 1 500 — 500 − 2 500 L-carvone 15 485 − 3 500 L-carvone 37.5462.5 − 4 500 L-carvone 75 425 − 5 500 L-carvone 112.5 387.5 − 6 500L-carvone 150 350 Sl+ 7 500 L-carvone 225 275 + 8 500 L-carvone 450 50 +9 500 Citral 15 485 − 10 500 Citral 37.5 462.5 − 11 500 Citral 75 425 −12 500 Citral 112.5 387.5 Sl+ 13 500 Citral 150 350 + 14 500 Citral 225275 + 15 500 Citral 450 50 +

As can be seen from the results in Table 9, YP is capable of absorbingand encapsulating terpenes at high YP concentration. YP absorbed andencapsulated at least 112.5 μl of L-carvone terpene emulsion or at least75 μl of citral emulsion per 125 mg of YP. This demonstrates that theterpene encapsulation reaction is independent of YP concentration withinthe ranges tested.

EXAMPLE 16 Screen Commercially Available Particles for TerpeneAbsorption

The following protocol was performed to analyse the loading propertiesof different types of particles. The particles studied were Baker'sYeast Particles (Sigma Chemical Corp., St. Louis, Mo.), Nutrex™ Walls(Sensient Technologies, Milwaukee, Wis.), SAF-Mannan™ (SAF Agri,Minneapolis, Minn.), Nutricept Walls™ (Nutricepts Inc., Burnsville,Minn.), Levacan™ (Savory Systems International, Inc., Branchburg, N.J.)and WGP™ (Alpha-beta Technology, Inc. Worcester, Mass.).

L-carvone and citral emulsions were prepared by sonicating 7 g terpene+3ml 3.3% Tween-80.

Table 24 below compares the purity with the number of yeast particlesper mg and the packed solids weight/volume ratio.

TABLE 24 Yeast Purity No. particles/ Mg particles/ Particle % Beta1,3-glucan mg ml Bakers 11.2  4 × 10⁷ 250 Nutrex 24.5 1.7 × 10⁸ 58.8 SAFMannan 33.4 2.4 × 10⁸ 41.7 2.7 × 10⁸ Nutricepts 55.7 5.2 × 10⁸ 37Levacan 74.6  1 × 10⁸ 19.2 WGP 82.1 3.5 × 10⁸ 10

From Table 24 it can be concluded that the number of particles per mg isinversely proportional to purity. Thus the number of particles per mg ofWGP is almost 10-fold higher than Baker's YP.

The YP suspensions were prepared as follows:

-   -   Baker's yeast particle suspension (YP) was prepared by mixing        250 mg YP/ml 1% Tween 80.    -   Nutrex suspension was prepared by mixing 163 mg Nutrex YGP/ml 1%        Tween 80.    -   SAF Mannan suspension was prepared by mixing 234 mg Biospringer        YGP/ml 1% Tween 80.    -   Nutricepts suspension was prepared by mixing 99 mg Nutricepts        YGP/ml 1% Tween 80.    -   Levacan suspension was prepared by mixing 217 mg Lev YGP/ml 1%        Tween 80.    -   WGP suspension was prepared by mixing 121 mg WGP YGP/ml 1% Tween        80.

The packed volume of the above particles is identical which means thatequal numbers of particles were assayed.

Loading reactions were set up as shown in Table 25 and left to incubateovernight. Samples were centrifuged at 14,000×g for 10 minutes and theappearance of free terpene floating on the aqueous layer and the colorof the encapsulated terpenes in the pellet was scored. The results areshown in the two right hand columns of Table 25. The highest volume ofterpene absorbed by particles as evidenced by the absence of freeterpene was recorded as the volume of absorbed terpene emulsion.

TABLE 25 conc Terpene Vol 1% Tween 80 Free Tube Particle mg/ml μlEmulsion μl μl Terpene Colour 1 Baker's 250 500 L-carvone 125 375 − W 2Nutrex 163 500 L-carvone 125 375 − W 3 SAF Mannan 234 500 L-carvone 125375 − W 4 Nutricepts 99 500 L-carvone 125 375 + W 5 Levacan 217 500L-carvone 125 375 + W 6 WGP 121 500 L-carvone 125 375 + W 7 Baker's 250500 Citral 100 375 − Y 8 Nutrex 163 500 Citral 100 375 − Y 9 SAF Mannan234 500 Citral 100 375 − W 10 Nutricepts 99 500 Citral 100 375 + Y 11Levacan 217 500 Citral 100 375 + int 12 WGP 121 500 Citral 100 375 + int13 — — — L-carvone 125 875 + — 14 — — — Citral 100 900 + Y W = white; Y= yellow; sl = slight; int = intermediate

From the results the following conclusions were reached:

-   -   Purified particles with a low lipid content were less effective        at absorbing terpenes.    -   Less pure particles were more effective at absorbing terpenes.    -   Yellow degradation product of citral was not formed when        encapsulated in SAF-Mannan™.    -   Based on qualitative loading at the single terpene level tested,        SAF Mannan™ appears to be best, Nutrex™ second and Baker's        third.

EXAMPLE 17 Kinetics of Terpene Loading into Various Types of Particlesand Different Incubation Temperatures

The following protocol was adopted to compare the loading kinetics ofvarious types of yeast particles.

L-carvone and citral emulsions were prepared by sonicating 7 g terpenewith 3 ml 3.3% Tween-80.

1% Tween-80 solution was prepared by sonicating 1 ml 10% Tween-80 in 10ml water.

-   -   Baker's YP was prepared by mixing 5 g of bakers YP in 20 ml 1%        Tween-80.    -   Nutrex™ YGP suspension was prepared by mixing 2 g Nutrex™ YGP in        20 ml 1% Tween-80.    -   SAF Mannan™ suspension was prepared by mixing 2 g SAF Mannan™ in        20 ml 1% Tween-80.

Loading reactions were set up as shown in Table 26.

The reactions were incubated for 1, 3, 6, 9 and 24 hours at roomtemperature or 37° C. After incubation samples were centrifuged at14,000×g for 10 minutes and the appearance of free terpene floating onthe aqueous layer was scored. The results are shown in the two righthand columns of Table 26. The highest volume of terpene absorbed by theparticles as evidenced by the absence of free terpene was recorded asthe volume of absorbed terpene emulsion. Colour of the encapsulatedpellet was scored at 24 hours.

TABLE 26 T conc Terpene Vol Free Terpene (hr) Tube ° C. Particle mg/mlμl Emulsion μl 1% Tween-80 1 3 6 9 24 Color 1 Rt Bakers 250 3500L-carvone 788 2712 + − − − − W 2 37 Bakers 250 3500 L-carvone 788 2712 +− − − − W 3 Rt Nutrex 100 3500 L-carvone 1050 2450 + − − − − W 4 37Nutrex 100 3500 L-carvone 1050 2450 + − − − − W 5 Rt SAF 100 3500L-carvone 1050 2450 <+  − − − − W 6 37 SAF 100 3500 L-carvone 1050 2450<+  − − − − W 7 Rt Bakers 250 3500 Citral 525 2975 + − − − − Y 8 37Bakers 250 3500 Citral 525 2975 + − − − − VY 9 Rt Nutrex 100 3500 Citral788 2712 + − − − − Y 10 37 Nutrex 100 3500 Citral 788 2712 + − − − − VY11 Rt SAF 100 3500 Citral 788 2712 + − − − − W 12 37 SAF 100 3500 Citral788 2712 + − − − − W White, W; Yellow, Y; Very Yellow, VY; RoomTemperature, Rt

From the results shown in Table 26 and other observations the followingconclusions can be made:

-   -   Terpene loading reaction takes between 1 and 3 hours.    -   Terpene loading occurs faster at 37° C. than at room        temperature.    -   SAF Mannan™ appears to be preferable particles for two reasons:        -   Faster and more complete uptake of both terpenes.        -   Citral remains stable when loaded as evidenced by the            absence of yellow colour, characteristic of citral            degradation, after 24 hours at 37° C.

EXAMPLE 18 Screen a Range of Single Terpenes and Terpene Combinationsfor Particle Loading

The following protocol was adopted to compare the loading efficiency ofBaker's YP versus SAF Mannan™.

Terpene emulsions were prepared as follows:

-   -   L-carvone—4.5 g L-carvone in 1.5 ml 3.3% Tween-80.    -   Citral—4.5 g citral in 1.5 ml 3.3% Tween-80.    -   Thymol/L-carvone mixture (T/L)—2.25 g thymol and 2.25 g        L-carvone in 1.5 ml 3.3% Tween-80.    -   Eugenol—4.5 g eugenol in 1.5 ml 3.3% Tween-80.    -   Geraniol—4.5 g geraniol in 1.5 ml 3.3% Tween-80.    -   Citral/L-carvone/Eugenol mixture (C/L/E)—1.5 g citral, 1.5 g        L-carvone, 1.5 g eugenol in in 1.5 ml 3.3% Tween-80.

Emulsions composed of terpene:water:surfactant ratio of 0.75:0.3:0.05were used for these experiments.

Increasing volumes of terpene emulsion were mixed with 250 mg/ml Baker'sYP or 250 mg/ml SAF Mannan™ overnight at room temperature as shown inTables 27 and 28. Samples were centrifuged at 14,000×g for 10 minutesand the appearance of free terpene floating on the aqueous layer wasscored. The highest volume of terpene emulsion absorbed by Baker's YP orSAF Mannan™ as evidenced by the absence of free terpene was recorded asthe volume of absorbed terpene emulsion. Colour of encapsulated terpenesin the pellet was recorded. The results in Tables 27 and 28 show thatall single and terpene combinations were efficiently loaded into bothBaker's YP or SAF Mannan particles.

TABLE 27 Evaluation of Baker's YP Loading of Different Terpenes andTerpene Mixtures. Baker Terpene Vol 1% Tween-80 Free Tube (μl) Emulsion(μl) (μl) Terpene Colour 1 500 — — 500 − W 2 500 L-carvone 15 485 − W 3500 L-carvone 37.5 462.5 − W 4 500 L-carvone 7 425 +/− W 5 500 L-carvone112.5 387.5 +/− W 6 500 L-carvone 150 350 + W 7 500 L-carvone 225 275 +W 8 500 L-carvone 450 50 ++ W 9 500 Citral 15 485 − Y 10 500 Citral 37.5462.5 − Y 11 500 Citral 75 425 − Y 12 500 Citral 112.5 387.5 +/− Y 13500 Citral 150 350 + Y 14 500 Citral 225 275 + Y 15 500 Citral 450 50 +Y 16 500 T/L 15 485 − W 17 500 T/L 37.5 462.5 − W 18 500 T/L 75 425 − W19 500 T/L 112.5 387.5 +/− W 20 500 T/L 150 350 + W 21 500 T/L 225 275 +W 22 500 T/L 450 50 + W 23 500 Eugenol 15 485 − W 24 500 Eugenol 37.5462.5 − W 25 500 Eugenol 75 425 − W 26 500 Eugenol 112.5 387.5 +/− W 27500 Eugenol 150 350 + W 28 500 Eugenol 225 275 + W 29 500 Eugenol 45050 + W 30 500 Geraniol 15 485 − W 31 500 Geraniol 37.5 462.5 − W 32 500Geraniol 75 425 − W 33 500 Geraniol 112.5 387.5 + W 34 500 Geraniol 150350 + W 35 500 Geraniol 225 275 + W 36 500 Geraniol 450 50 + W 37 500C/L/E 15 485 − Y 38 500 C/L/E 37.5 462.5 − Y 39 500 C/L/E 75 425 − Y 40500 C/L/E 112.5 387.5 +/− Y 41 500 C/L/E 150 350 + Y 42 500 C/L/E 225275 + Y 43 500 C/L/E 450 50 + Y

TABLE 28 Evaluation of SAF Mannan Loading of Different Terpenes andTerpene Mixtures. SAF Terpene 1% Tween-80 Free Tube (μl) Emulsion Vol(μl) Terpene Colour 1 500 — — 500 − W 2 500 L-carvone 15 485 − W 3 500L-carvone 37.5 462.5 − W 4 500 L-carvone 75 425 − W 5 500 L-carvone112.5 387.5 − W 6 500 L-carvone 150 350 +/− W 7 500 L-carvone 225 275+/− W 8 500 L-carvone 450 50 + W 9 500 Citral 15 485 − W 10 500 Citral37.5 462.5 − W 11 500 Citral 75 ul 425 − W 12 500 Citral 112.5 387.5 − W13 500 Citral 150 350 +/− W Inverted 14 500 Citral 225 275 + W Inverted15 500 Citral 450 50 + W Inverted 16 500 T/L 15 485 − W 17 500 T/L 37.5462.5 − W 18 500 T/L 75 425 − W 19 500 T/L 112.5 387.5 − W 20 500 T/L150 350 +/− W 21 500 T/L 225 275 + W 22 500 T/L 450 50 + W 23 500Eugenol 15 485 − W 24 500 Eugenol 37.5 462.5 − W 25 500 Eugenol 75 425 −W 26 500 Eugenol 112.5 387.5 +/− W 27 500 Eugenol 150 350 + W 28 500Eugenol 225 275 + W 29 500 Eugenol 450 50 + W 30 500 Geraniol 15 485 − W31 500 Geraniol 37.5 462.5 − W 32 500 Geraniol 75 425 − W 33 500Geraniol 112.5 387.5 − W 34 500 Geraniol 150 350 − W 35 500 Geraniol 225275 − W Inverted 36 500 Geraniol 450 50 + W Inverted 37 500 C/L/E 15 485− W 38 500 C/L/E 37.5 462.5 − W 39 500 C/L/E 75 425 − W 40 500 C/L/E112.5 387.5 − W 41 500 C/L/E 150 350 − W 42 500 C/L/E 225 275 +/− W 43500 C/L/E 450 50 + W Inverted = Phase Inverted - solids floating ontop - no free oil; W = white; Y = yellow.

From the results the following observations were made:

-   -   All terpenes appeared to load into Baker's YP and SAF Mannan.    -   SAF Mannan has a higher terpene loading capacity than bakers YP.    -   The two and three way mixtures of terpenes also appear to        efficiently load.    -   The terpene Eugenol appears to have a higher density than the        particles and water as it was found associated with the pellet.    -   For the SAF Mannan, the higher load levels and lighter particles        resulted in loaded particles floating on the surface of the        aqueous layer for citral and geraniol.    -   Citral was protected from oxidation by the SAF Mannan but not by        the Baker's YP.

The approximate maximal loading for each particle type was determinedand is shown in tables 29 and 30 below. Percentage loaded represents aratio of the amount of terpene loaded to the amount of particle present(weight for weight).

TABLE 29 Maximal terpene loading in Baker's YP. Terpene Vol. Loaded μl %Loaded w/w L-carvone 37.5 33.3 Citral 75 67% Thymol/L-carvone 1:1 75 67%Eugenol 75 67% Geraniol 75 67% Citral/L-carvone/ 75 67% Eugenol (1:1:1)

TABLE 30 Maximal terpene loading in SAF Mannan. Terpene Vol. loaded μl %Loaded w/w L-carvone 112.5 100% Citral 150 133% Thymol/L-carvone 1:1112.5 100% Eugenol 112.5 100% Geraniol 150 133% Citral/L-carvone/ 150133% Eugenol (1:1:1)

EXAMPLE 19 Evaluation of Terpene Stability in Aqueous Emulsions andEncapsulated Terpene Formulations

Terpene stability was assessed by the observation of citral formulationsfor the formation of a yellow colored oxidation product. As noted in theright hand column in Tables 25-28 citral emulsions and citralencapsulated Bakers YP turned a progressively increasing yellow colorover time. However, citral encapsulation in SAF Mannan™ increased citralstability as evidenced by a reduction or absence of yellow color overtime.

EXAMPLE 20 Loading of Terpenes in Minimal Water

The following protocol was carried out to evaluate the possibility thatterpene loading and encapsulation into YP could be carried out at a veryhigh Yeast Particles (YP) solids level to allow for direct extrusion ofthe loaded formulation into a fluidised bed drier. The minimal amount ofwater to completely hydrate the SAF Mannan™ particles was determined tobe 3.53 g water per g solids. This defines the hydrodynamic volume (HV)or water absorptive capacity of the particles. At this level of waterthe hydrated particles have a consistency of a stiff dough which isthixotropic, i.e. shear thinning like mayonnaise. Addition of water upto 40% above the HV results in a thick flowable paste. The standardreaction that has been used in the above examples was carried out at3×HV water.

A series of terpene (L-carvone) loading reactions were carried outkeeping the ratio of particle:terpene:Tween (1:0.44:0.04) constant andvarying the amount of water in the system from the HV (3.53 g) to HV+40%water (4.92 g). Controls were the standard loading system which uses3×HV water, particles only and terpene only reactions. Followingovernight incubation samples of the mixtures were evaluatedmicroscopically for free terpene and evidence of terpene uptake into theparticles and for material flow characteristics by assessing flow ininverted tubes over 15 minutes. In addition, the presence of free oilwas assessed by hydrating the reaction mixture with 5×HV, vortexing toobtain a complete dispersion of particles and centrifugation to sedimentthe particle encapsulated terpene. The results are shown in Table 31 andFIGS. 7 to 12. FIGS. 7 to 12 show the loading results of the followingtubes:

-   -   FIG. 7—Tube 3    -   FIG. 8—Tube 5    -   FIG. 9—Tube 6    -   FIG. 10—Tube 8    -   FIG. 11—Tube 10    -   FIG. 12—Tube 11

TABLE 31 SAF Terpene Weight Water Free Tube g Emulsion (g) (g) TerpeneFlow 1 — L-carvone 4.64 4.5 + + 2 1 — — 8.0 − + 3 1 L-carvone 4.64 4.5− + 4 1 L-carvone 4.64 — − − 5 1 L-carvone 4.64 0.17 − − 6 1 L-carvone4.64 0.35 − − 7 1 L-carvone 4.64 0.52 − Sl 8 1 L-carvone 4.64 0.7 − Mod9 1 L-carvone 4.64 0.87 − High 10 1 L-carvone 4.64 1.05 − High 11 1L-carvone 4.64 1.39 − High

The results shown in Table 31 and FIGS. 7 to 12 demonstrate that terpeneloading and encapsulation into the particles occurred at all waterratios evaluated. Surprisingly, equivalent loading occurred even whenthe loading reaction was taking place in a reaction with the consistencyof a stiff dough using the minimal amount of water to hydrate theparticles. The absence of free terpene was observed microscopically(FIGS. 7 to 12) and in the low level of terpene in the supernatants, asevidenced by a marked reduction in the turbidity of the supernatantcompared to the terpene only control.

These results extend our understanding of the conditions to loadterpenes into hollow glucan particles. The flexibility to use a minimalvolume of water to hydrate the particles during the loading process willallow loading of the terpenes under conditions where the reactionmixture is a malleable dough-like consistency using standard food-gradeswept surface dough mixers. The consistency of the final high solidsterpene loaded mixture is suitable for direct extrusion to form noodlesand pellets for fluidised bed drying.

Suitable facilities to scale up production in this manner would require:

-   -   Gaulin homogeniser, or equivalent to produce stable terpene        emulsion.    -   Swept surface dough mixing tank.    -   Extruder.    -   Fluidised bed drier.

EXAMPLE 21 Evaluation of an Interstitial Hydrocolloid Agent to AidDispersion in Dried Hollow Glucan Particles Encapsulating a TerpeneComponent Dispersion when Re-Hydrated

The following protocol was adopted to evaluate the effect of aninterstitial hydrocolloid to increase dried hollow glucan particleencapsulated terpene formulations to disperse when hydrated.

-   -   SAF Mannan™ particles    -   0.1% Tween 80    -   L-carvone    -   Xanthan Gum—1% w/v in 0.1% Tween 80

The effect of increasing xanthan gum levels on dry hollow glucanparticle encapsulated L-carvone dispersion in water was assessed byloading L-carvone into SAF Mannan by incubating 1.1 g of an L-carvoneemulsion (L-carvone:water:surfactant ratio of 0.75:0.3:0.05) with 1 gSAF Mannan and 4.4 g 0.1% Tween 80 containing 0-1% xanthan gum as shownin Table 32.

TABLE 32 L-carvone 0.1% 1% Emulsion Tween-80 Xanthan Visual Tube SAF g(g) (g) (g) Observations 1 1 1.1 4.4 0 Large non- uniform clumps 2 1 1.14.33 0.07 Uniform suspension 3 1 1.1 4.26 0.14 Uniform suspension 4 11.1 4.12 0.28 Uniform suspension 5 1 1.1 3.85 0.55 Uniform suspension 61 1.1 3.3 1.1 Finer Uniform suspension 7 1 1.1 2.2 2.2 Finer Uniformsuspension 8 1 1.1 0 4.4 Finer Uniform suspension

The results in Table 32 and FIGS. 13 to 20 demonstrate that theinclusion of a high molecular weight hydrocolloid during the drying ofthe particle encapsulated terpene aids in the hydration and dispersionof the microparticles into a uniform suspension. Other examples of suchhydrocolloid agents are maltodextrin, alginates, or the like.

It may also be worthwhile to include a pellet coating to increase thestability of the loaded terpenes, and to provide a sustained release ofterpene.

EXAMPLE 22 Nematocidal Activity of Encapsulated Terpenes

Preparations of yeast cell walls encapsulating citral were preparedaccording to the procedures described above. The hollow glucan particlescontained 17.5% citral, and the particles were present at in the testpreparations at a concentration of 1000 ppm. This means that terpeneswere effectively present at a concentration of 175 ppm.

1.0 ml of the test preparations was added to 0.1 to 0.15 ml of watercontaining root-knot nematodes. 1.0 water was added to the nematodes asthe control.

Observations were made as [revopis;u described and the kill rateassessed (i.e. percentage dead) after 24 and 48 hrs. The results shownbelow in Table 13 are an average of 2 sets of results.

TABLE 33 Nematicidal activity of encapsulated terpene solution (17.5%citral @ 1000 ppm) Kill Rate Time Test Control 24 h 45 17 48 h 56 21

The results demonstrate that hollow glucan particles encapsulatingterpenes are effective at killing root-knot nematodes at a particleconcentration of 1000 ppm, which corresponds to a citral concentrationof only 175 ppm.

Thus hollow glucan particles encapsulating terpenes appear to be aseffective as terpenes in solution or with surfactant as nematicides. Thenematicidal activity is retained despite the terpene being encapsulatedwithin the particle. It can be expected that higher concentrations ofterpenes within the hollow glucan particles, or higher concentrations ofthe particles would result in an even higher kill rate, as is the casefor terpenes in solution or with surfactant.

The invention claimed is:
 1. A nematicidal composition comprising ahollow glucan particle encapsulating a terpene component, wherein theterpene component is an active agent, and wherein the hollow glucanparticle has a β-1,3-glucan content from 24% to 35% w/w.
 2. Thenematicidal composition according to claim 1, wherein the nematicidalcomposition further comprises water.
 3. The nematicidal compositionaccording to claim 1, wherein the terpene component comprises one ormore terpenes selected from the group consisting of citral, pinene,nerol, b-ionone, geraniol, carvacrol, eugenol, carvone, terpeniol,anethole, camphor, menthol, limonene, nerolidol, farnesol, phytol,carotene (vitamin A), squalene, thymol, tocotrienol, perillyl alcohol,borneol, myrcene, simene, carene, terpenene, and linalool.
 4. Thenematicidal composition according to claim 1, wherein the terpenecomponent is a combination of thymol and geraniol.
 5. The nematicidalcomposition according to claim 1, wherein the nematicidal compositioncomprises the terpene component at a concentration of from 125 ppm to2000 ppm in water.
 6. The nematicidal composition according to claim 1,wherein the hollow glucan particles are yeast cell walls.
 7. Thenematicidal composition according to claim 6, wherein the hollow glucanparticles are obtained from the insoluble waste stream of a yeastextract manufacturing process.
 8. The nematicidal composition accordingclaim 1, wherein the hollow glucan particles have a lipid contentgreater than 5% w/w.
 9. The nematicidal composition according to claim1, wherein the hollow glucan particles encapsulating the terpenecomponent comprises 10% to 67% w/w terpene component, 0.1% to 10%surfactant, and 40% to 90% hollow glucan particles.
 10. A compositioncomprising a hollow glucan particle encapsulating a terpene component,wherein the terpene component is an active agent, and wherein the hollowglucan particle has a β-1,3-glucan content from 24% to 35% w/w.
 11. Thecomposition according to claim 10, wherein the composition furthercomprises water.
 12. The composition according to claim 10, wherein theterpene component comprises one or more terpenes selected from the groupconsisting of citral, pinene, nerol, b-ionone, geraniol, carvacrol,eugenol, carvone, terpeniol, anethole, camphor, menthol, limonene,nerolidol, farnesol, phytol, carotene (vitamin A), squalene, thymol,tocotrienol, perillyl alcohol, borneol, myrcene, simene, carene,terpenene, and linalool.
 13. The composition according to claim 10,wherein the terpene component is a combination of thymol and geraniol.14. The composition according to claim 10, wherein the compositioncomprises the terpene component at a concentration of from 125 ppm to2000 ppm in water.
 15. The composition according to claim 10, whereinthe hollow glucan particles are yeast cell walls.
 16. The compositionaccording to claim 15, wherein the hollow glucan particles are obtainedfrom the insoluble waste stream of a yeast extract manufacturingprocess.
 17. The composition according claim 10, wherein the hollowglucan particles have a lipid content greater than 5% w/w.
 18. Thecomposition according to claim 10, wherein the hollow glucan particlesencapsulating the terpene component comprises 10% to 67% w/w terpenecomponent, 0.1% to 10% surfactant, and 40% to 90% hollow glucanparticles.
 19. The nematicidal composition according to claim 1, whereinthe nematicidal composition further comprises a surfactant.
 20. Thenematicidal composition according to claim 10, wherein the nematicidalcomposition further comprises a surfactant.